Serotonin 5-HT2c receptors have been implicated in several neurologic conditions such as schizophrenia, overeating, depression, and epileptic- like seizures. However, it is unknown when and where 5-HT2C receptors function in the brain. Since 5-HT2C receptors are phosphorylated upon exposure to agonists, we will generate antibodies against phosphorylated epitopes to probe activation of 5-HT2C receptors in vivo. Specific aims are designed to: 1) identify phosphorylated sites of 5-HT2C receptors. Phospho-epitope mapping experiments will be performed with 5-HT2C receptor antibodies to identify potential phosphorylated regions of receptors expressed in NIH/3T3 cells. Deletion mutants lacking various regions of 5-HT2C receptors will be examined in gel-shift and phosphate-incorporation assays to identify phosphorylated regions. Wild-type receptors will be subjected to phosphoamino analyses to identity phosphorylated amino acids. Phospho-epitope mapping experiments will be performed with native receptors to test the hypothesis that native receptors are phosphorylated in the same region as recombinant receptors. The results from mutant receptors and phosphoamino analyses of wild-type receptors will be used to design site-directed mutants that lack potential phosphorylation sites. Incorporation of phosphate in the site-directed mutants will be measured to identify sites required for agonist-mediated phosphorylation of 5-HT2C receptors. 2) generate and characterize anti- (phospho)-peptide antibodies. Antibodies will be generated against phosphorylated peptides that correspond to the region(s) of 5-HT2C receptors sensitive to agonist-mediated phosphorylation. Once specificity of antibodies against the phosphorylated form of 5-HT2C receptors has been established in cell lines, reactivity of antibodies against native 5-HT2C receptors will be evaluated on immunoblots. The anti-(phospho)-peptide antibodies generated during this research period will be powerful tools to probe activation of 5-HT2C receptors in vivo. The long-term goal of this research is to address the role of 5-HT2C receptors in epilepsy and hallucinogenic drug activity.